compositions of buffers are given in Table A5 of the Appendix. The protein concentration was estimated using Bradford assay.
3.3 Protein estimation by Bradford’s assay:
The concentration of purified protein samples (RDT or MBP-peptides) were estimated by Bradford assay (Sigma) (164). Amount of protein adsorbed on NPs was determined by Bradford assay according to a protocol used by Kockbek et al. (165). Coated or uncoated NPs were resuspended in 200 μl of PBS. Bradford reagent (200 µl) was added to each sample and incubated for 10 minutes at room temperature. Subsequently, absorbance was measured at 595 nm. Solutions of BSA with known concentrations were prepared to generate a standard curve.
The amount of protein attached to NPs was estimated by using standard graph.
3.4 Mammalian cell culture:
Human glioblastoma cell lines, U-87 MG; mouse macrophage cell line, RAW 264.7; human breast cell line, MDA-MB 468 were procured from NCCS, Pune, India. These cells were cultured in DMEM growth medium with high glucose (HiMedia) supplemented with 10% fetal bovine serum (Gibco) and antibiotics (Anti-anti, Gibco). The cells were sub-cultured by trypsinization once it reached 80-90% confluency. The adherent cells were washed with sterile PBS (pH 7.4) and detached with 0.25% Trypsin-EDTA (Gibco). After a few minutes, trypsin was removed out of the flask and cells were flushed with the fresh medium to make a uniform cell suspension.
Cells were centrifuged at 2,400 rpm for 6 min at 4 ˚C. The pellet was resuspended in 1 ml complete media. Cells density was estimated using trypan blue dye exclusion procedure. The dead cells don‟t take dye, but viable cells do. 10 µl of resuspended cells were mixed with 10 µl of 0.4% trypan blue (Invitrogen). Then, 10 µl of mixed sample is loaded in cell counting chamber slide (Invitrogen). This slide was inserted in automated cell countess (Invitrogen). Total numbers of cells, including viable and dead cells, were recorded. The desire cell density was reseeded on another 25 cm2 flask or 6 or 96 well plates. The cells were maintained in humidified 5% CO2
incubator at 37 °C.
The cells were also cryopreserved for a longer period in freezing media. Freezing media is composed of 95% serum and 5% dimethyl sulfoxide (DMSO). DMSO acts as cryoprotectant which minimizes cell damage occur during the freezing process. Cells were resuspended in 1 ml freezing media and placed in liquid nitrogen (-196 ˚C) for longer storage. Medium and other reagents used in mammalian cell culture are given in Table A4 in the Appendix.
3.5 Synthesis of poly (lactic-co-glycolic acid) PLGA Nanoparticles:
The synthesis of poly (lactic-co-glycolic acid) PLGA nanoparticle (NPs) was carried out by using previously developed emulsification solvent evaporation method (166). 5 mg of PLGA was dissolved in either 1 ml dichloromethane (DCM, Merck) or 1 ml chloroform (Merck). Different solvent results in different sizes of nanoparticles. Further, PLGA solution was added dropwise in 0.5% Polyvinyl alcohol in water under continuous sonication at 30% amplitude for 2 min at room temperature (RT). Here, PVA was used as a non-ionic emulsifying agent. During synthesis, either dye or drug was loaded. Fluorescein or Rhodamine 123 (5 µM) was incorporated to make these nanoparticles fluorescent. Dye was dissolved in 0.5% PVA in water. An Anti-cancer drug, Irinotecan was used for the experiments. For drug-loaded nanoparticles, Irinotecan in water was mixed with PLGA solution and sonicated well to create a well formed emulsion. This was subsequently added dropwise to PVA solution. NPs were washed thrice with water to remove the excess solvent. Subsequently, it was dried by vacuum drying. The protein-coated NPs was formed by incubating recombinant receptor binding domain of DT (RDT, 100 µg/ml) in PBS (pH 7.4) with NPs (5 mg/ml) for 2 h at 37 ˚C. A schematic diagram of NP synthesis is given in Figure 3.1.
Figure 3.1: Synthesis of NPs. PLGA was dissolved either in dichloromethane or chloroform and added dropwise to 0.5% polyvinyl alcohol solution under continuous sonication at 30 Kpsi for 2 min at room temperature.
3.6 Transmission Electron Microscope (TEM):
The size and morphology of PLGA NPs (NPs) and RDT coated NP (RDT-NPs) were observed by Transmission Electron Microscope, (TEM; JEM 2100; Jeol). Sample of RDT-NPs was prepared by incubating NPs with RDT protein for 2 h and then centrifuged at 8,000 rpm for 5 min at RT. The supernatant was removed and pellet was diluted in distilled water. The samples were added in the carbon coated copper grid and kept for drying for 4 h at room temperature.
TEM was operated at an accelerating voltage of 200 KV. The size of NPs was calculated by ImageJ software (167).
3.7 Field Emission Scanning Electron Microscope (FESEM):
The size and surface morphology of NPs and RDT-NPs were observed by Field Emission Scanning Electron Microscope (FESEM; Zeiss, model sigma). The samples were diluted in water and drop cast into the aluminum foil wrapped coverslip and dried for overnight. Sputter coating is applied using ultra-thin gold coating over the polymeric nanoparticle. Sputter coating provides thermal conductivity to sample and reduce microscope beam damage. Samples were imaged with an accelerating voltage of 3 kV. The size of NPs was calculated by ImageJ software (167).
3.8 Dynamic light scattering (DLS):
The size of particles is determined by the random movement of particles in a liquid medium using dynamic light scattering (DLS; Zetasizer; Nano ZS, Malvern). NPs and RDT-NPs were resuspended in filtered water. The particle size, size distribution, polydispersity index (PDI) and zeta potential of NPs and RDT-NPs were analyzed using DLS.
3.9 UV-Visible absorption spectroscopy:
The association of RDT with nanoparticles (NPs) is detected using UV-Vis absorption spectroscopy. The fixed amount of NPs (500 µg) is incubated with different concentration of recombinant RDT (0-25 µg/ml) in PBS (pH 7.4) for 2 h. Then, NPs were centrifuged at 8,000 rpm for 5 min. The pellet having NPs coated with RDT (RDT-NPs) was dissolved in 1 ml water.
The absorbance of RDT-NPs is measured at 280 nm. Water is used as a blank solution and its reading was subtracted from samples.
The absorbance spectrum of Irinotecan was measured using UV-Visible spectrophotometer (Beckman coulter, DU 730). 10 µg/ml of Irinotecan dissolved in water. The spectrum was recorded from 210 to 600 nm using 10 mm pathlength quartz cuvette.
3.10 Fourier transform infrared spectroscopy (FT-IR):
FT-IR spectroscopy was used for characterization of NPs and protein-coated NPs. The NPs and RDT-NPs were mixed separately with IR grade, potassium bromide (Sigma) in the ratio of 1:100. Pellets were prepared by applying 5.5 metric ton pressure with a hydraulic press. The infrared absorption spectra were collected in an inert atmosphere over a wave number range of 450-4000 cm-1 in FT-IR instrument (Perkin Elmer; Spectrum Two).
3.11 ELISA to detect extent of coating of NPs by RDT:
The coating of recombinant RDT on NPs was detected by ELISA. Recombinant RDT has His- tag. It was detected using anti-His antibody. NPs were incubated with varying concentration of RDT in PBS for 2 h at 37 ˚C. Subsequently, NPs were separated by centrifugation and resuspended in 1 ml PBS. 100 µl of resuspended NPs was taken in 1.5 mL tubes and separated by centrifugation. The supernatant was discarded. NPs were resuspended and incubated in blocking buffer (2% BSA in PBS), for 2 h at room temperature. Subsequently, NPs were separated by centrifugation, resuspended, and washed with PBS. These NPs were resuspended in 100 μL of 2% BSA in PBS (pH 7.4) with mouse anti-His antibody (1:1000 dilution, Calbiochem) and incubated for 1 h at room temperature. NPs were collected by centrifugation, washed with PBS, and resuspended in 100 μl of HRP-conjugated anti-mouse antibody (1:1000 dilution, Sigma-Aldrich) in 2% BSA-PBS. After 1 h of incubation at room temperature, NPs were harvested by centrifugation, washed with PBS, and resuspended in 400 μL of o-Phenylenediamine dihydrochloride (OPD) substrate. After a few minutes of incubation at room temperature, 100 μL of the sample was transferred to wells of ELISA plate. Reaction was stopped by adding 50 μL of 8 N H2SO4, and absorbance was measured at 492 nm. The composition of substrate solution is given in table A5 (Appendix).
Stability of RDT adsorbed on NPs was also determined by ELISA. For this, RDT-coated NPs were resuspended in serum-free media and incubated for different time durations at 37 °C.
Subsequently, NPs were separated by centrifugation and resuspended in 1 ml PBS. 100 µl of resuspended NPs was taken in 1.5 mL tubes and separated by centrifugation. The supernatant was discarded. NPs were resuspended and incubated in blocking buffer (2% BSA in PBS), for 2
h at room temperature. Subsequently, NPs were separated by centrifugation, resuspended, and washed with PBS. These NPs were resuspended in 100 μl of 2% BSA in PBS (pH 7.4) with mouse anti-His antibody (1:1000 dilution, Calbiochem) and incubated for 1 h at room temperature. NPs were collected by centrifugation, washed with PBS, and resuspended in 100 μl of HRP-conjugated anti-mouse antibody (1:1000 dilution, Sigma-Aldrich) in 2% BSA-PBS.
After 1 h of incubation at room temperature, NPs were harvested by centrifugation, washed with PBS, and resuspended in 400 μl of o-Phenylenediamine dihydrochloride (OPD) substrate. After few minutes of incubation at room temperature, 100 μl of sample was transferred to wells of ELISA plate. Reaction was stopped by adding 50 μl of 8 N H2SO4, and absorbance was measured at 492 nm. The composition of substrate solution is given in table A5 (Appendix).
3.12 RNA isolation:
Total RNA was isolated from mammalian cell line for the preparation of cDNA. Total RNA is extracted by TRI reagent (Sigma). 1 ml of TRI reagent was added in 25 cm2 culture flasks having 1 x 106 cells. The cells were pooled and passed several times through a syringe to form a homogeneous lysate. The samples were kept at room temperature for 5 min to ensure complete dissociation of nucleoprotein complexes. Then, 200 µl of chloroform per ml of TRI reagent was added. After, 30-60 s of vigorous shaking, sample was allowed to stand for 10 min at room temperature. This mixture was then centrifuged at 12,000 g for 15 min at 4 ˚C. The RNA present in upper aqueous phase was transferred to fresh tube. 500 µl of 2-propanol is added to the tube and mix properly. Samples were kept for 10 min in room temperature. Then, it was centrifuged at 12,000 rpm for 10 min at 4 ˚C. Supernatant was removed and RNA pellet washed with 1ml 75% ethanol. Then, samples were centrifuged at 12,000 rpm for 10 min at 4 ˚C.
Supernatant was removed and RNA pellet was air-dried. Finally, RNA pellet was resuspended in water (20-30 µl)
3.13 Synthesis of cDNA:
The cDNA is prepared using verso cDNA synthesis kit (Thermoscientific). Reverse transcriptase is an RNA dependent DNA polymerase with attenuated RNase activity. It synthesizes long cDNA strand up to 11 kb. 1 µg of total RNA (10 µl) is heated at 65 ˚C for 5 min in a thermal cycler (Bio-Rad, C1000). This step will remove all the secondary structure present in RNA.
Then, 10 µl master mix was added to the tube. The mixture of total RNA and master mix is again placed in a thermal cycler for 60 min at 42 ˚C. The enzyme was inactivated at 95 ˚C for 2 min. The composition of master mix is given below:
Components Volume
5X cDNA synthesis Buffer 4 µl
Verso Enzyme mix 1 µl
Random Hexamer (500 ng/µl) 1 µl
dNTPs (5 mM each) 2 µl
Water 2 µl
Total volume 10 µl
3.14 RT-PCR (Reverse-transcriptase PCR)
The expression of HB-EGF in U 87-MG cells was determined using RT-PCR. The cDNA prepared from U 87-MG cells is used as a template. The HB-EGF specific primer is used and primer detail is given in table A7 of Appendix. The composition of master mix and PCR program are given below:
Components Volume
cDNA 1 µl
2X Master mix 10 µl
HBEGF FP (200nM) 1 µl
HBEGF RP (200nM) 1 µl
Water 7 µl
Total volume 20 µl
PCR program:
3.15 Spectrofluorometer:
The leaching of dye, Rhodamine 123 from NPs was checked by Spectrofluorometer. This experiment was used to assess the extent of leaching of the dye with time. PLGA NPs with Rhodamine 123 or without the dye were resuspended in serum-free culture media and
incubated at 37 ˚C, for different durations, with mild shaking. Subsequently, NPs were separated by centrifugation and resuspended in PBS. Fluorescent intensity of these NPs were measured by spectrofluorimetry with pathlength of 1 cm, excitation wavelength of 505 nm (Slit width = 1 nm) and emission wavelength of 528 nm (Slit width = 5 nm).
The mechanism of RDT-NPs internalization is elucidated using Spectrofluorometer. The U-87 MG cells were seeded on T-25 flask at a density 2 x 105 cells/flask. The cells were treated with different endocytosis inhibitors. After 2 h treatment, cells were washed with PBS twice and then trypsinized. Cells were resuspended in 1 ml serum media and then centrifuged at 3,000 rpm for 7 min at room temperature. The pellet was collected and washed with PBS followed by centrifugation. The pellet was resuspended in 200 µl PBS. The cells were lysed by using ultrasonicator (Hielscher) at amplitude 20 % for 10 s. The cell lysate were further diluted in PBS and fluorescence intensity of Rhodamine 123 was measured by Fluoromax-4 (Horiba scientific) with λEx = 505 nm, λEm = 528 nm, Pathlength = 1 cm. The slit width of excitation and emission were 1 and 5 nm, respectively.
The uptake of Transferrin-FITC was also measured using Spectrofluorometer. The U 87-MG cells were treated with transferrin-FITC (0.1 µM and 0.25 µM) along with or without chlorpromazine (5 µM). The cell lysate was prepared as above. The fluorescence intensity of Rhodamine 123 was measured by Fluoromax-4 (Horiba scientific) with λEx = 492 nm, λEm = 518 nm, Pathlength = 1 cm. The slit width of excitation and emission were 1 and 5 nm, respectively.
3.16 Flow cytometer:
The uptake of NPs was also determined by flow cytometer. The U-87 MG or RAW 264.7 cells were seeded in 6 well plates (1 x 105 cells/well) and maintained in humidified 5% CO2 incubator at 37 ˚C. After 48 h incubation, cells were treated with uncoated NPs; BSA- or RDT-coated NPs in serum-free medium for 2 h at 37 ˚C. After trypsinization, cells were harvested by centrifugation and re-suspended in 1 ml Serum free media. The fluorescence intensity of Rhodamine 123 was measured by flow cytometer in FL-1 channel (FACSCalibur, BD, USA) and data analyzed by FCS-express software.
3.17 Fluorescence Imaging:
The U-87 MG (1 x 105) cells were seeded over cover slip in each 35 mm petri plate having 2 ml complete media. Cells were incubated for 48 h in humidified 5% CO2 incubator at 37 ˚C. Cells were treated with different sample in serum-free medium for 2 h. After treatment, media was
removed and cells were washed with PBS. The nuclear staining was done by using 4',6- diamidino-2-phenylindole (DAPI). DAPI (4 µg/ml in PBS) was added to the cells and incubated for 5 min at room temperature. Excess of stain was removed by multiple washing with PBS. The cells were fixed with 3.7% formaldehyde in PBS for 20 minutes at room temperature. After washing with PBS, cells were imaged using inverted Epi-fluorescence microscope (Eclipse TiU, Nikon). The uptake of Rhodamine 123 loaded NPs were detected using a filter for green fluorescence (B-2E/C, Nikon) and DAPI using (UV-2E/C, Nikon). The composition of 3.7%
formaldehyde is given in table A5 of the appendix.
3.18 Cell viability assay:
Cell viability was estimated by MTT assay (168). The U 87-MG cells (104/well) or MDA MB 468 cells (5 x 103/well) were seeded in 96 well plates. The cells were cultured for 48 h at 37 °C in 5% CO2. Later, cells were treated with different sample for a specific duration in serum-free medium. Subsequently, 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) solution (10 µl, 5 mg/ml in PBS) was added to each well. Further, Plate was incubated for 2 h at 37 °C in 5% CO2, wrapped in aluminum foil. After incubation, MTT-containing medium was replaced by 100 µl dimethyl sulfoxide (DMSO, Merck) to solubilize MTT-formazan crystals. After incubation for 10 min at 37°C, absorbance was measured at 570 nm and reference reading at 690 nm was recorded by a microplate reader (Infinite 200 PRO, Tecan). Cell viability (%) was measured with respect to untreated cells.
3.19 In-vitro drug release assay:
During NP synthesis, 1.65 mg of Irinotecan was loaded in 2.5 mg PLGA NP. The Irinotecan- loaded NPs (2.5 mg) were dispersed in 1 ml of PBS (pH 7.4) and incubated at 37 ˚C with gentle shaking. At different time point, Irinotecan-loaded NPs was harvested by centrifugation at 10, 000 rpm for 5 min at room temperature. Release of Irinotecan was assayed by measuring absorbance of supernatant at 255 nm and 370 nm. A standard curve was made by measuring absorbance of Irinotecan solution of different concentrations. Release of the drug percentage from NPs was calculated from the absorbance data using the standard curve.
3.20 Synthesis of peptides:
200 mg of Rink amide MBHA resin (loading 0.7 mmol) was taken with a syringe containing frit and was allowed to swell with DCM solvent first and then with DMF solvent. After that, Fmoc group of the resin was cleaved with 20% pip/DMF. The resin was then washed with DMF solvent. Then, 2.0 equivalent of Fmoc-Gly-OH, 2.5 equivalent of o-NosylOXY (coupling reagent) and 4.5 equivalent of DIPEA (base) were dissolved in DMF solvent and added to the syringe.
The syringe was allowed to rotate at SPPS rotator for coupling. After that, the reaction mixture in the syringe was washed with DMF and DCM. It was then acetylated (capping) with 2 equivalent of Ac2O, 2 equivalent of NMI in DCM solvent. Again Fmoc cleavage of the amino acid attached with resin was carried out with 20% pip/DMF and the peptide sequence was continued in the same procedure said above. At last, Fmoc group was cleaved with 20%
pip/DMF. Finally, cocktail cleavage was carried out with 2ml of TFA: DCM (8.5:1.5) and one drop of H2O for 5h to cleave C-terminus of the peptide from rink amide resin. After completion of reaction, the mixture was precipitated in cold diethyl ether solvent to get the crude peptide.
3.21 Liquid chromatography:
Crude peptides were dissolved in Acetonitrile/Water (1:1) and purified by RP-HPLC (Waters 600E) using a C18-μ Bondapak column at a flow rate of 4 mL/ min. Binary solvent system was used, solvent A (0.1 % Trifluoroacetate in H2O) and solvent B (0.1 % Trifluoroacetate in Acetonitrile). A Waters 2489 UV detector was used with an option of dual detection at 214 and 254 nm. A total run time of 20 min. was given and gradient used for purification was 5−100 % acetonitrile for 18 min followed by 100% acetonitrile till 20 min.
The purity of the peptide DTP28 was confirmed using Waters 600E Analytical HPLC system, Ascentis C18 analytical column at a flow rate of 1 ml/min, linear gradient of 5-100% acetonitrile over 18 minutes in a total run time of 20 min. Dual wavelength was selected at 214 nm and 254 nm. The purity of the other three peptides, DTP28A, DTP28AE and DTP28Neg were confirmed using Agilent-Q-TOF 6500 instrument, in ESI-LCMS positive mode, Agilent eclipse plus C18 analytical column at a flow rate of 0.3 ml/min, linear gradient of 5-100% CH3CN over 6 minutes in a total run time of 7 min. Wavelength was selected at 214 nm.
3.22 Mass spectrometry:
The mass of the peptide samples were analyzed on Agilent-Q-TOF 6500 instrument, in electrospray ionization (ESI) positive mode, equipped with the Mass hunter work station software.
MALDI mass of the peptide samples were analyzed using CHCA matrix on BRUKER autoflex speed instrument which consists of a MALDI ionization source for samples in the solid state and a TOF/TOF mass analyzer, equipped with Bruker daltonics flex analysis software. To prepare CHCA matrix, 1.0 mg of CHCA was dissolved in 100 μl of CH3CN/H2O (1:1) containing 0.1%
TFA.
3.23 Tricine SDS-PAGE:
Tricine SDS-PAGE is commonly used to separate protein in the mass range 1-100 kDa. The purity of short peptides of molecular weight ~2.9 kDa is checked by tricine SDS-PAGE. Tricine SDS-PAGE was run according to the protocol of Hermann Schagger (169). Briefly, an acrylamide gel was prepared by introducing a 10% 'spacer gel' between 4% stacking and 16%
separating gels to sharpen the peptide bands. Anode buffer as the lower electrode buffer and cathode buffer as the upper electrode buffer were used in vertical electrophoresis apparatus.
The gel was run at constant voltage of 30 V until sample crosses the stacking gel and then voltage was increased to 90 V and run up to the end. Peptides were visualized by silver staining. Buffer compositions of Tricine SDS-PAGE are given in Appendix (Table A5).
3.24 Silver staining:
The Tricine SDS-PAGE gel was stained by silver staining. Protein detection using silver staining is 50 times higher sensitive than coomassie brilliant blue staining. Tricine SDS-PAGE gel was incubated in fixing solution for 30 min. Subsequently, incubation solution was added and incubated for 30 min. Then the gel was washed with distilled water for 3 times (5 min each).
Silver staining solution was added and incubated for 50 min. Gel was developed using developing solution. Once bands appear, stop solution was added. The gel image was taken by gel documentation system (Bio-Rad, ChemiDoc XRS+). The Buffer compositions of silver staining are given in Appendix (Table A5).